U.S. patent number 10,345,298 [Application Number 15/306,344] was granted by the patent office on 2019-07-09 for diagnosis of multiple sclerosis.
This patent grant is currently assigned to Klinikum rechts der Isar der Technischen Universitat Munchen. The grantee listed for this patent is Klinikum rechts der Isar der Technischen Universitat Munchen. Invention is credited to Bernhard Hemmer, Lucas Schirmer, Rajneesh Srivastava.
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United States Patent |
10,345,298 |
Hemmer , et al. |
July 9, 2019 |
Diagnosis of multiple sclerosis
Abstract
The present invention relates to a method for diagnosing
multiple sclerosis (MS) and/or clinically isolated syndrome (CIS)
and/or radiologically isolated syndrome (RIS) or a predisposition
for either condition in a subject, the method comprising
determining the presence of an anti-KIR4.1 antibody in a sample
obtained from said subject by (a) contacting said sample with a
protein; and (b) detecting the formation of a protein-anti-KIR4.1
antibody complex; wherein said protein is KIR4.1, wherein
glycosylation of the large extracellular domain of said KIR4.1 is
as in human oligodendrocytes or glycosylation of the large
extracellular domain is absent; and wherein the formation of said
complex is indicative of MS, CIS, RIS or a predisposition
therefore. Furthermore provided is an antibody or fragment or
derivative thereof competing with the anti-KIR4.1 antibody in a
sample obtained from a patient having MS.
Inventors: |
Hemmer; Bernhard (Munchen,
DE), Srivastava; Rajneesh (Munchen, DE),
Schirmer; Lucas (Munchen, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
Klinikum rechts der Isar der Technischen Universitat
Munchen |
Munchen |
N/A |
DE |
|
|
Assignee: |
Klinikum rechts der Isar der
Technischen Universitat Munchen (Munchen, DE)
|
Family
ID: |
50677983 |
Appl.
No.: |
15/306,344 |
Filed: |
April 30, 2015 |
PCT
Filed: |
April 30, 2015 |
PCT No.: |
PCT/EP2015/059531 |
371(c)(1),(2),(4) Date: |
October 24, 2016 |
PCT
Pub. No.: |
WO2015/166057 |
PCT
Pub. Date: |
November 05, 2015 |
Prior Publication Data
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|
|
Document
Identifier |
Publication Date |
|
US 20170074877 A1 |
Mar 16, 2017 |
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Foreign Application Priority Data
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|
|
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Apr 30, 2014 [EP] |
|
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14166705 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/564 (20130101); G01N 2800/285 (20130101) |
Current International
Class: |
G01N
33/564 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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239 400 |
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Aug 1994 |
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EP |
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2530088 |
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Dec 2012 |
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EP |
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WO 89/09622 |
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Oct 1989 |
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WO |
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WO 91/10741 |
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Jul 1991 |
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WO |
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WO 94/02602 |
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Feb 1994 |
|
WO |
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WO 96/33735 |
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Oct 1996 |
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WO |
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WO 96/34096 |
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Oct 1996 |
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WO |
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WO 02/18650 |
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Mar 2002 |
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WO |
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WO 2012/163765 |
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Dec 2012 |
|
WO |
|
Other References
N Eng. J. Med 2012 vol. 367, p. 115-123 The Supplementary Appendix
total 30 pages (Year: 2012). cited by examiner .
Ascherio et al., "Environmental Risk Factors for Multiple
Sclerosis. Part I: The Role of Infection", Ann. Neurol., vol. 61,
No. 4, pp. 288-299, 2007. cited by applicant .
Ascherio et al., "Environmental Risk Factors for Multiple
Sclerosis. Part II: Noninfectious Factors", Ann. Neurol., vol. 61,
No. 6, pp. 504-513, 2007. cited by applicant .
Galfre et al., "Preparation of Monoclonal Antibodies: Strategies
and Procedures", Methods in Enzymology, vol. 73, 45 pages, 1981.
cited by applicant .
Hafler et al., "Risk Alleles for Multiple Sclerosis Identified by a
Genomewide Study," The New England Journal of Medicine, vol. 357,
No. 9, pp. 851-862, 2007. cited by applicant .
Jung et al., "Lines of Murine Oligodendroglial Precursor Cells
Immortalized by an Activated neu Tyrosine Kinase Show Distinct
Degrees of Interaction with Axons In Vitro and In Vivo," European
Journal of Neuroscience, vol. 7, pp. 1245-1265, 1995. cited by
applicant .
Kohler et al., "Continuous Cultures of Fused Cells Secreting
Antibody of Predefined Specificity", Nature, vol. 256, pp. 495-497,
1975. cited by applicant .
Malmborg et al., "BIAcore as a Tool in Antibody Engineering,"
Journal of Immunological Methods, vol. 183, pp. 7-13, 1995. cited
by applicant .
McFarland et al., "Multiple Schlerosis: A Complicated Picture of
Autoimmunity", Nature Immunology, vol. 8, pp. 913-919, 2007. cited
by applicant .
Noseworthy et al., "Multiple Schlerosis", The New England Journal
of Medicine, vol. 343, pp. 938-952, 2000. cited by applicant .
PCT International Search Report and Written Opinion for
PCT/EP2015/059531 dated Jun. 22, 2015 (12 pages). cited by
applicant .
Schier et al., "Efficient in vitro Affinity Maturation of Phage
Antibodies Using BIAcore Guided Selections", Human Antibodies
Hybridomas, vol. 7, pp. 97-105, 1996. cited by applicant .
Srivastava et al., "Potassium Channel KIR4.1 as an Immune Target in
Multiple Sclerosis", New England Journal of Medicine, vol. 367, No.
2, pp. 115-123, Jul. 12, 2012. cited by applicant .
Thrower et al., "Clinically Isolated Syndromes", Neurology, vol.
68, pp. S12-S15, 2007. cited by applicant.
|
Primary Examiner: Cheu; Changhwa J
Attorney, Agent or Firm: Mueting, Raasch & Gebhardt,
P.A.
Claims
The invention claimed is:
1. A method for detecting a polypeptide-anti-KIR4.1 complex, the
method comprising: (a) contacting a sample obtained from a subject
with a polypeptide; and (b) detecting a polypeptide anti-KIR4.1
antibody complex; wherein the polypeptide comprises: glycosylated
KIR4.1, wherein glycosylation of the large extracellular domain of
said KIR4.1 is as in human oligodendrocytes; or a subsequence of
human KIR4.1 comprising SEQ ID NO: 1 or 2, wherein the N residue of
SEQ ID NO: 1 or 2 is glycosylated as the corresponding N residue of
the large extracellular domain of KIR4.1 of human
oligodendrocytes.
2. The method of claim 1, wherein the polypeptide is a component of
an in vitro cell.
3. The method of claim 1, wherein one or more of the following
applies: (a) KIR4.1 is human KIR4.1; (b) said polypeptide is a
homotetramer of KIR4.1; (c) KIR4.1 consists of the amino acid
sequence of SEQ ID NO: 3; (d) said extracellular domain consists of
the amino acid sequence of SEQ ID NO: 4; and (e) glycosylation of
the entire protein is as in human oligodendrocytes.
4. The method of claim 1, wherein the polypeptide is obtained from
human oligodendrocytes or from a cell line of oligodendrocytic
origin.
5. The method of claim 2, wherein the polypeptide is in the cell
membrane of the in vitro cell.
Description
This application is a U.S. National Stage Application of
International Application No. PCT/EP2015/059531, filed Apr. 30,
2015, which was published in English on Nov. 5, 2015, as
International Publication No. WO 2015/166057 A1. International
Application No. PCT/EP2015/059531 claims priority to European
Application No. 14166705.5, filed Apr. 30, 2014.
The present invention relates to a method for diagnosing multiple
sclerosis (MS), clinically isolated syndrome (CIS) and/or
radiologically isolated syndrome (RIS) or a predisposition for
either condition in a subject, the method comprising determining
the presence of an anti-KIR4.1 antibody in a sample obtained from
said subject by (a) contacting said sample with a protein or
peptide; and (b) detecting the formation of a protein-anti-KIR4.1
antibody complex or a peptide-anti-KIR4.1 antibody complex,
respectively; wherein said protein is KIR4.1, wherein glycosylation
of the large extracellular domain of said KIR4.1 is as in human
oligodendrocytes or glycosylation of the large extracellular domain
is absent; wherein said peptide (i) consists of a subsequence of
the large extracellular domain of human KIR4.1, (ii) which
subsequence is at least 5 consecutive amino acid residues long,
(iii) which subsequence comprises or consists of SEQ ID NO: 1 or 2,
and (iv) wherein the residue N of SEQ ID NO: 1 or 2 is glycosylated
as the corresponding N residue of the large extracellular domain of
KIR4.1 of human oligodendrocytes or is not glycosylated; and
wherein the formation of said complex is indicative of MS, CIS, RIS
or a predisposition therefore.
In this specification, a number of documents including patent
applications and manufacturer's manuals is cited. The disclosure of
these documents, while not considered relevant for the
patentability of this invention, is herewith incorporated by
reference in its entirety. More specifically, all referenced
documents are incorporated by reference to the same extent as if
each individual document was specifically and individually
indicated to be incorporated by reference.
Multiple sclerosis (MS) is the most common chronic inflammatory
disease of the central nervous system (CNS) leading to disability
in the majority of affected patients (Noseworthy et al., N. Engl.
J. Med. 343, 938-952 (2000)). The etiology of MS is unknown but
epidemiological evidence suggests a complex interplay between
genetic and environmental factors (Ascherio et al., Ann. Neurol.
61, 504-513 (2007); Ascherio et al., Ann. Neurol. 61, 288-299
(2007); Hafler et al., N. Engl. J. Med. 357, 851-862 (2007)). An
uncertain pathogenic mechanism, clinical heterogeneity and
unpredictability of the outcome of individual patients add to the
complexity of the disease (McFarland et al., Nat. Immunol. 8,
913-919 (2007)).
In WO 2012/163765, the present inventors disclosed that KIR4.1, a
specific inward rectifying potassium channel is involved in the
etiology of multiple sclerosis. More specifically, a subset of
multiple sclerosis patients has anti-KIR4.1 autoantibodies in its
serum.
In the present application, the present inventors investigated
further into possibilities for sensitively detecting said
autoantibodies. In doing so, the inventors aimed to provide
improved means and methods for the diagnosis of multiple sclerosis.
In particular, it turned out that the presence vs. absence and in
particular nature of glycosylation of KIR4.1 influences antibody
binding.
In a first aspect, the present invention relates to a method for
diagnosing multiple sclerosis (MS), clinically isolated syndrome
(CIS) and/or radiologically isolated syndrome (RIS) or a
predisposition for either condition in a subject, the method
comprising determining the presence of an anti-KIR4.1 antibody in a
sample obtained from said subject by (a) contacting said sample
with a protein or peptide; and (b) detecting the formation of a
protein-anti-KIR4.1 antibody complex or a peptide-anti-KIR4.1
antibody complex, respectively; wherein said protein is KIR4.1,
wherein glycosylation of the large extracellular domain of said
KIR4.1 is as in human oligodendrocytes or glycosylation of the
large extracellular domain is absent; wherein said peptide (i)
consists of a subsequence of the large extracellular domain of
human KIR4.1, (ii) which subsequence is at least 5 consecutive
amino acid residues long, (iii) which subsequence comprises or
consists of SEQ ID NO: 1 or 2, and (iv) wherein the residue N of
SEQ ID NO: 1 or 2 is glycosylated as the corresponding N residue of
the large extracellular domain of KIR4.1 of human oligodendrocytes
or is not glycosylated; and wherein the formation of said complex
is indicative of MS, CIS, RIS or a predisposition therefore.
In a preferred embodiment glycosylation of the large extracellular
domain of said KIR4.1 is as in human oligodendrocytes.
The term "peptide" refers to a polycondensate of amino acids.
Preferably, said amino acids are selected from the 20 naturally
occurring amino acids. The upper length limit of the peptide of the
present invention is given by the limitation that the peptide is a
subsequence of the large extracellular domain of KIR4.1, wherein,
in a preferred embodiment, the length of said extracellular domain
is defined by the length of SEQ ID NO: 4.
Said subsequence which is at least 5 consecutive amino acid
residues long is preferably at least 6, at least 7, at least 8, at
least 9, at least 10, at least 11, at least 12, at least 13, at
least 14 or at least 15 consecutive amino acid residues long. A
preferred subsequence which is exactly 15 consecutive amino acid
residues long is the sequence of SEQ ID NO: 2. In further preferred
embodiments, said subsequence has a length of at least 20, at least
25, at least 30 or at least 35 consecutive amino acid residues.
Said subsequence may also consist of or comprise any one of SEQ ID
NOs: 7 to 14. Subsequences comprising any one of SEQ ID NOs: 7 to
14 include subsequences consisting of a sequence of any one of SEQ
ID NOs: 7 to 14 wherein either His or His-Thr is added to the
C-terminus.
A further preferred subsequence is that from residue 10 to residue
15 of SEQ ID NO: 2, herewith enclosed as SEQ ID NO: 16
(PPANHT).
In any of the above-defined peptides, 1 or more, preferably, 2, 3,
4, 5, 6, 7, 8, 9 or 10 amino acids may be replaced by other amino
acids, provided that the sequence of SEQ ID NO: 1, which is
comprised in any of the above-defined peptides, remains unaltered.
To the extent any of the above-defined peptides comprises the
sequence of SEQ ID NO: 2 in its entirety, it is preferred that the
sequence of SEQ ID NO: 2 in its entirety remains unaltered.
The term "protein" has its art-established meaning. In particular,
a protein can comprise one or more polypeptides. A polypeptide is a
polycondensate of amino acids, wherein no particular length limit
applies. A preferred protein in accordance with the present
invention is a homotetramer. Proteins, even if they are monomeric,
may differ from mere polycondensates of amino acids in that one or
more posttranslational modifications are present. A key feature of
the present invention is the posttranslational modification which
is glycosylation. While the specific requirements of the present
invention in terms of glycosylation are defined herein above and
below, it is noted that further posttranslational modifications may
be present or absent. Having regard to the protein of the present
invention which is KIR4.1, it is preferred that in the large
extracellular domain thereof there is exactly one site which is a
target of posttranslational modification. Said posttranslational
modification is N-glycosylation, and it concerns a specific Asn
residue as defined above.
It is noted that both protein and peptide according to the present
invention are characterized in that they are capable of binding to
the anti-KIR4.1 antibody which is present in the serum of a
subgroup of multiple sclerosis patients. This antibody is also
referred to as "autoantibody" herein. This autoantibody preferably
recognizes an epitope comprising or consisting of SEQ ID NO: 17
(PPAN) which is comprised within SEQ ID NO: 2.
Said autoantibody is to be distinguished from a further antibody
which is an antibody according to one of the aspects of the present
invention detailed further below. The latter antibody is capable of
competing with said autoantibody for binding to the same epitope in
the large extracellular domain of KIR4.1.
Provided with the teaching of the present invention and that of the
present inventors' earlier application WO 2012/163765, said
autoantibody can be pulled down from the serum of patients having
MS and purified. Suitable means for precipitating the antibody
include the receptor and peptide as defined in WO 2012/163765 as
well as the proteins and peptides according to the present
invention.
The term "multiple sclerosis" refers to an inflammatory disease
affecting the nervous system; see also the literature quoted in the
background section above. Whether or not a subject or patient has
multiple sclerosis can be determined with the method of diagnosing
according to the invention. Alternatively or in addition, a
diagnosis of multiple sclerosis can be established on the basis of
established clinical symptoms, said clinical symptoms being known
to the skilled person. The clinical symptoms of multiple sclerosis
include vision problems, dizziness, vertigo, sensory dysfunction,
weakness, problems with coordination, loss of balance, fatigue,
pain, neurocognitive deficits, mental health deficits, bladder
dysfunction, bowel dysfunction, sexual dysfunction, heat
sensitivity. Diagnosis on the basis of said clinical symptoms,
however, will generally be less sensitive as compared to the
methods of the present invention. Moreover, in many cases
predisposition will be characterized by the absence of any clinical
symptoms in which case diagnosis of predisposition can only be
effected by using the method of the present invention or, in the
alternative, that of the present inventors' earlier invention
described in WO 2012/163765.
CIS is generally perceived in the art as being an early stage MS,
wherein the clinical parameters characteristic of the latter are
not yet fully developed. For a discussion of CIS, see, for example,
Thrower, Neurology 68, S12-S15 (2007).
Analogously, also radiologically isolated syndrome (RIS) is
considered an early stage MS. RIS patients are patients who
received an MRI of the brain for non-MS related reasons. The
patient has MS-like lesions although the patient has not yet
classical MS symptoms. These patients are diagnosed with RIS. They
have an about 30% chance to develop MS within the next years.
The present inventors detected in earlier studies that high titers
of anti-KIR4.1 autoantibodies are found in the serum of 50.8% of
patients. Accordingly, the presence of anti-KIR4.1 autoantibodies
defines a subgroup of MS patients. It is expected that this
subgroup responds or responds particularly well to treatment with
peptides according to the present invention. Determining whether a
given individual belongs to this subgroup can be determined with
the diagnostic means and methods as disclosed herein as well as in
WO 2012/163765.
The method of the first aspect permits to diagnose multiple
sclerosis, or, to the extent multiple sclerosis is not apparent in
said subject, for diagnosing a predisposition therefore. The term
"predisposition" has the meaning as established in the art and
prefers a likelihood to develop a disease. In particular, said
likelihood is higher than in a normal control subject. Said
likelihood in a normal control subject may be represented as the
average likelihood to develop MS in a random sample from the
population.
A preferred group of individuals to be tested for said
predisposition are individuals with a history of MS in the
family.
Given that the present inventors detected high titers of
anti-KIR4.1 antibodies in sera of a significant fraction of MS
patients, the means and methods described herein allow diagnosis of
MS or a predisposition therefore in about half of the MS cases or
subjects being at risk to develop the disease, respectively. In
particular, the methods of the invention permit early diagnosis of
MS or a predisposition therefore or a confirmation of an uncertain
diagnosis. The antibody test may allow to diagnose CIS or MS
without invasive procedures (such as cerebrospinal fluid analysis)
and to diagnose MS, CIS or predisposition to MS earlier than this
would be possible by diagnostic procedures known in the art. It is
well known that MS therapy works best when started as early as
possible during the course of disease. Therefore, early diagnosis
may allow to implement early treatment of patients with CIS, MS or
at risk to develop these diseases. In some individuals at risk
treatment may even prevent the (further) development of
disease.
Said contacting is to be effected under conditions allowing the
formation of the complex defined in item (b) of the first aspect.
Exemplary or preferred conditions are described in the Examples
enclosed herewith; see the section "KIR4.1 ELISA", as well as
further below.
Detecting the formation of said complex can be done with
art-established methods. Basically, methods for detecting the
formation of a complex comprising an antigen and an antibody are
applicable for that purpose. Preferred methods are detailed further
below.
The present inventors discovered that, in the sequence of the large
surface domain of KIR4.1, the epitope recognized by the anti-KIR4.1
autoantibody as present in the sera of MS patients includes a site
for N-glycosylation. Furthermore, it turned out that presence vs.
absence and in particular nature of glycosylation of the mentioned
site influences antibody binding. In particular, high degrees of
glycosylation caused less sensitive detection or may abrogate
antibody binding altogether. A "high degree of glycosylation"
refers to a large oligosaccharide moiety being bound. An exemplary
highly glycosylated form has an apparent molecular weight of about
49 to about 55 kD on a denaturing gel. Therefore, for the assay for
anti-KIR4.1 autoantibodies to be sensitive, the probe, i.e. the
protein or peptide of the present invention, may be
non-glycosylated. The non-glycosylated form of KIR4.1 from humans
has an apparent molecular weight of about 34 kD on a denaturing
gel. If non-glycosylated forms are to be obtained from glycosylated
forms, this is preferably done by treatment with the enzyme
peptide-N-glycosidase (PNGase).
Preferably, KIR4.1 and the protein of the invention is glycosylated
only to a low degree, more specifically it is glycosylated as in
human oligodendrocytes. The term "glycosylated as in human
oligodendrocytes" refers to the presence of an oligosaccharide
moiety which is identical or substantially identical to the
oligosaccharide moiety which is present on human KIR4.1 as it
occurs in human oligodendrocytes. The glycosylation site of human
KIR4.1 is defined by SEQ ID NO: 1 or 2, more specifically by the
N-residue thereof, noting that SEQ ID NO: 1 or 2 is a subsequence
of the large extracellular domain of human KIR4.1. The N residue is
comprised in a NXT motif for N glycosylation, residue X being H in
case of KIR4.1. The sequence NHT forms the last three residues of
SEQ ID NO: 16. On a denaturing gel, KIR4.1, when glycosylated as in
human oligodendrocytes, has an apparent molecular weight of about
38 to about 42 kD, more preferably from about 40 to about 42 kD.
Especially preferred is an apparent molecular weight of about 42
kD. The term "apparent molecular weight" refers, as usual, to the
molecular weight on a denaturing gel.
While short peptides, when manufactured synthetically, will not be
glycosylated, this does not apply for KIR4.1 as well as fragments
thereof which are obtained from natural sources including cell
lines. To the extent peptides and proteins from natural sources are
to be used, it is therefore of utmost importance to pay attention
to the degree of glycosylation.
To the extent glycosylation of said protein and said peptide is as
in human oligodendrocytes, it is preferred that at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98% and at least 99% of said protein or peptide is glycosylated as
in human oligodendrocytes. More specifically, the large
extracellular domain of said protein is glycosylated as in human
oligodendrocytes, and the above-defined residue N is glycosylated
as in human oligodendrocytes. Analogously, and to the extent use is
to be made of non-glycosylated forms of said protein or said
peptide, it is preferred that at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98% and at least
99% is non-glycosylated, more specifically non-glycosylated in the
large extracellular domain in case said protein is to be used, and
non-glycosylated at the above-defined N residue in case use is to
be made of said peptide. Having said that, it is also envisaged to
use mixturesconsisting of or comprising glycosylated and
non-glycosylated forms, glycosylated forms being glycosylated as in
human oligodendrocytes. To the extent such mixture is to be used,
it is understood that preferably at least 80%, at least 85%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98% and at least
99% of said mixture exclusively consist of the mentioned
glycosylated and non-glycosylated forms.
Under physiological conditions, KIR4.1 occurs as a homotetramer. To
the extent the assay in accordance with the first aspect of the
present invention makes use of said protein, said protein
preferably is in tetrameric form. Preferably, said peptide is in
tetrameric form. Preference is given to those tetrameric forms of
said peptide which mimic the tetrameric arrangement of epitopes as
provided by the naturally occurring KIR4.1 homotetramer. Preferred
epitopes are those defined by peptides comprising or consisting of
SEQ ID NOs: 1 or 2. Envisaged is also the use of the monomeric form
as well as of other oligomeric forms such as dimers, trimers,
pentamers, hexamers etc, noting that the use of higher order
aggregates is less preferred.
In a preferred embodiment, said anti-KIR4.1 antibody is present in
said sample, (a) presence of at least one clinical symptom of MS,
CIS or RIS in said subject is indicative of MS, CIS or RIS,
respectively; and (b) absence of any clinical symptom of MS, CIS
and RIS is indicative of said predisposition for MS, CIS and
RIS.
As disclosed above, the methods according to the invention provide
for diagnosing multiple sclerosis as well as for diagnosing a
predisposition therefor. The present preferred embodiment provides
for further information to be acquired for said subject, said
further information aiding in distinguishing between diagnosis of
the disease and diagnosis of a predisposition therefor. In
particular, said further information consists of or comprises at
least one clinical symptom of multiple sclerosis. Multiple
sclerosis is a well-known disease with established clinical
symptoms. The skilled person is well aware of clinical symptoms
being characteristic or indicative of multiple sclerosis (see also
above and below) and can determine the presence or absence thereof
without further ado.
In accordance with the present preferred embodiment, the absence of
any clinical symptom of multiple sclerosis, when concomitantly
occurring together with the presence of anti-KIR4.1 antibodies, is
indicative of predisposition for multiple sclerosis. In other
words, where established methods of diagnosis or prognosis fail,
the present invention allows to identify those subjects which
exhibit an elevated risk of developing multiple sclerosis at some
point in the future.
On the other hand, in subjects where at least one clinical symptom
of multiple sclerosis is present, the determination of anti-KIR4.1
antibodies further corroborates the diagnosis of multiple
sclerosis. In those cases where the clinical parameters alone do
not permit a clear diagnosis, the present invention aids in
performing and substantiating said diagnosis. This applies in
particular to early forms of multiple sclerosis. As is well-known
in the art, an early diagnosis of multiple sclerosis is highly
desirable, given that early stages are generally more amenable to
treatment.
According to a further preferred embodiment, said clinical symptom
is at least one selected from vision problems, dizziness, vertigo,
sensory dysfunction, weakness, problems with coordination, loss of
balance, fatigue, pain, neurocognitive deficits, mental health
deficits, bladder dysfunction, bowel dysfunction, sexual
dysfunction, heat sensitivity, the presence of (an) inflammation
marker(s) in cerebrospinal fluid (CSF), the presence of lesions of
the brain and/or the spinal cord. The mentioned lesions may be
detected in an MRT image. Typically, such lesions occur in the
periventricular, juxtacortical and/or infratentorial region of the
brain. Inflammation markers indicative of MS are well-known in the
art and are preferably to be selected from pleocytosis (abnormally
increased number of cells in the CSF, wherein typical values of
increased cell numbers are between 5 and 50 cells/.mu.l or above),
intrathecal IgG synthesis and the occurrence of oligoclonal IgG
bands in the CSF.
In a further preferred embodiment, the detection of the anti-KIR4.1
antibody or said complex, respectively, in said sample is effected
by a method selected from the group consisting of ELISA,
immunoprecipitation, Western blotting, immunofluorescence,
immunohistochemistry, flow cytometry, metalloimmunoassay (such as
GLORIA), fluorescence resonance energy transfer (FRET) assay and
mass spectroscopy. These methods are well-established and at the
skilled person's disposal. ELISA is particularly preferred. In an
ELISA assay, an antibody binding to said anti-KIR4.1 antibody may
be used. Similar considerations apply to immunoprecipitation,
immunofluorescence and immunohistochemistry. As noted above, the
skilled person, when provided with the teaching of the present
invention, can isolate and characterize the anti-KIR4.1 antibody
without further ado. Such characterization preferably uses mass
spectrometry. Once being characterized, mass spectrometry may be
used for determining presence or absence of anti-KIR4.1 antibodies
in any given sample. FRET assays may be used, for example, in the
context of a binding assay, said binding assay preferably making
use of a receptor, said receptor being defined further below. Such
FRET assay may be designed such that a detectable transfer between
donor and acceptor of the FRET pair only occurs in case receptor
and anti-KIR4.1 antibody are in close special proximity, said close
special proximity being indicative of the presence of the
anti-KIR4.1 antibody.
In a further preferred embodiment, said sample is selected from
blood, serum, plasma, lymph nodes, CSF, lacrimal fluid, urine,
sputum and brain biopsy.
In a further preferred embodiment, said protein or peptide is
comprised in an in vitro cell, preferably in the cell membrane of
said cell. In other words, it is preferred that the diagnostic
assay in accordance with the first aspect of the present invention
is implemented as a cell-based assay. It is especially preferred
that said cells have the protein or peptide in accordance with the
present invention incorporated into their cell membrane. This
provides for the epitope present in said protein or peptide to be
recognized and bound by the autoantibodies which are present in the
sera of MS patients. It is preferred that said cell is also used to
express the protein or peptide in accordance with the present
invention.
In a second aspect, the present invention provides a protein
wherein said protein is KIR4.1, and wherein glycosylation of the
large extracellular domain of said KIR4.1 is as in human
oligodendrocytes or absent.
The method according to the first aspect as disclosed herein above
provides for the use of a protein or peptide, respectively, wherein
both protein and peptide may be either non-glycosylated, in
particular non-glycosylated at the Asn residue comprised in SEQ ID
NO: 1 or 2, or may be glycosylated, in particular at said Asn in a
manner which is identical or substantially identical to the
glycosylation observed on human KIR4.1 in human oligodendrocytes.
Preferred is the protein, and particularly preferred is the protein
having a glycosylation status as in human oligodendrocytes. This is
the subject-matter of the second aspect. We note that the
glycosylation status in human oligodendrocytes, in particular at
said Asn residue, is unique. The experimental data comprised in the
examples provide further characterization of the glycosylation
status of KIR4.1 as it is found in human oligodendrocytes, in
particular in terms of the apparent molecular weight on a
denaturing gel.
In a preferred embodiment of the method according to the first
aspect as well as of the protein according to the second aspect,
one or more of the following applies: (a) KIR4.1 is human KIR4.1;
(b) said protein is a homotetramer of KIR4.1; (c) KIR4.1 consists
of the amino acid sequence of SEQ ID NO: 3; (d) said extracellular
domain consists of the amino acid sequence of SEQ ID NO: 4; and (e)
glycosylation of the entire protein is as in human
oligodendrocytes.
Human KIR4.1 occurs in its natural environment, including human
oligodendrocytes, in a homotetrameric form. The sequence of human
KIR4.1 is given in SEQ ID NO: 3. The sequence of the large
extracellular domain thereof is given in SEQ ID NO: 4.
In a third aspect, the present invention provides a peptide (i)
consisting of a subsequence of the large extracellular domain of
human KIR4.1, (ii) which subsequence is at least 5 consecutive
amino acid residues long, (iii) which subsequence comprises or
consists of SEQ ID NO: 1 or 2, and (iv) wherein the residue N of
SEQ ID NO: 1 or 2 is glycosylated as the corresponding N residue of
the large extracellular domain of KIR4.1 of human
oligodendrocytes.
As noted above, said peptide is an alternative to said protein. In
particular, it is envisaged that said peptide mimics said protein.
The Examples enclosed herewith provide a proof of principle that
the salient features of said protein which are key for the method
of diagnosis of the invention are indeed reproduced by said
peptide.
In any of the above-defined peptides, 1 or more, preferably, 2, 3,
4, 5, 6, 7, 8, 9 or 10 amino acids may be replaced by other amino
acids, provided that the sequence of SEQ ID NO: 1, which is
comprised in any of the above-defined peptides, remains unaltered.
To the extent any of the above-defined peptides comprises the
sequence of SEQ ID NO: 2 in its entirety, it is preferred that the
sequence of SEQ ID NO: 2 in its entirety remains unaltered.
We note that the present inventors' earlier application WO
2012/163765 also describes peptides, including peptides related to
the large extracellular domain of human KIR4.1. The specific
contribution of the present invention, however, is the recognition
of the specific requirements of the assay in terms of glycosylation
of the peptide for the assay to perform optimal. This is reflected
by the third aspect of the present invention.
In a preferred embodiment of all above disclosed aspects of the
present invention, said protein or peptide is obtained from human
oligodendrocytes or from a cell line of oligodendrocytic origin
such as Oli-neu oligodendrocytes.
Oligodendrocytes, also known as oligodendroglia, are cells of the
central nervous system of certain vertebrates including humans.
Their functions include the provision of support and insulation to
axons.
As an alternative to oligodendrocytes, cells or cell lines of
oligodendrocytic origin may be used as source of the protein or
peptide according to the present invention. A particularly
preferred cell line, said cell line being of oligodendrocytic
origin, are Oli-neu cells (Jung et al. European Journal of
Neuroscience, Vol. 7, pp. 1245-1265, 1995). Oli-neu cells as well
as other cells of oligodendrocytic origin are known or expected,
respectively, to provide the same type of N-linked glycosylation to
KIR4.1 as well as peptides derived therefrom, i.e., proteins and
peptides in accordance with the present invention. In a preferred
embodiment, proteins or peptides in accordance with the present
invention which are expressed from cells or cell lines of
oligodendrocytic origin are (i) isolated as a single peak,
preferably by means of gel filtration, which single peak
corresponds to the tetrameric form, and/or (ii) analyzed on a
denaturing gel in order to confirm that they have an apparent
molecular weight of about 38 to about 42 kDa. Lower molecular
weights may be acceptable as well.
In a fourth aspect, the present invention provides an antibody or
fragment or derivative thereof competing with the anti-KIR4.1
antibody in a sample obtained from a patient having MS.
The present inventors used the peptide of SEQ ID NO: 2 for the
purpose of raising an antibody. The most antigenic sequence within
SEQ ID NO: 2 is the sequence of SEQ ID NO: 1. This is a first
feature characterizing said antibody. A second feature
characterizing said antibody is its capability to compete with the
autoantibody. Furthermore, and similar to MS patients'
autoantibodies, said antibody preferably binds with a binding
affinity to KIR4.1 from human oligodendrocytes which binding
affinity is higher than the binding affinity for lower or higher
glycosylated forms. Said antibody is a useful constituent of the
kit according to the present invention, which kit is disclosed
further below.
As already noted above, it is preferred that said antibody binds to
a peptide consisting of SEQ ID NO:1 or 2, wherein said peptide is
either not glycosylated or glycosylated as the corresponding N
residue of the large extracellular domain of KIR4.1 of human
oligodendrocytes.
Antibodies as disclosed herein may be monoclonal or polyclonal
antibodies. Furthermore, the term "antibody" includes single chain
antibodies or fragments thereof that specifically bind to their
respective target as well as bispecific antibodies, synthetic
antibodies, antibody fragments such as Fab, F(ab.sub.2)', Fv and
scFv fragments and the like as well as chemically modified
derivatives thereof.
Monoclonal antibodies can be prepared, for example, by the
techniques as originally described in Kohler and Milstein, Nature
256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which
comprise the fusion of mouse myeloma cells to spleen cells derived
from immunized mammals with modifications developed by the art.
Furthermore, antibodies or fragments thereof to the aforementioned
peptides can be obtained by using methods which are described,
e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH
Press, Cold Spring Harbor, 1988. When derivatives of said
antibodies are obtained by the phage display technique, surface
plasmon resonance as employed in the BIAcore system can be used to
increase the efficiency of phage antibodies which bind to an
epitope of the peptide or polypeptide of the invention (Schier,
Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol.
Methods 183 (1995), 7-13). The production of chimeric antibodies is
described, for example, in WO89/09622. A further source of
antibodies to be utilized in accordance with the present invention
are so-called xenogenic antibodies. The general principle for the
production of xenogenic antibodies such as human antibodies in mice
is described in, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO
96/33735. Antibodies to be employed in accordance with the
invention or their corresponding immunoglobulin chain(s) can be
further modified using conventional techniques known in the art,
for example, by using amino acid deletion(s), insertion(s),
substitution(s), addition(s), and/or recombination(s) and/or any
other modification(s) known in the art either alone or in
combination. Methods for introducing such modifications in the DNA
sequence underlying the amino acid sequence of an immunoglobulin
chain are well known to the person skilled in the art; see, e.g.,
Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.
The term "monoclonal" or "polyclonal antibody" (see Harlow and
Lane, (1988), loc. cit.) also relates to derivatives of said
antibodies which retain or essentially retain their binding
specificity. Whereas particularly preferred embodiments of said
derivatives are specified further herein below, other preferred
derivatives of such antibodies are chimeric antibodies comprising,
for example, a mouse or rat variable region and a human constant
region.
The term "scFv fragment" (single-chain Fv fragment) is well
understood in the art and preferred due to its small size and the
possibility to recombinantly produce such fragments.
In a particularly preferred embodiment of the method of the
invention, said antibody or antibody binding portion is or is
derived from a human antibody or a humanized antibody. The term
"humanized antibody" means, in accordance with the present
invention, an antibody of non-human origin, where at least one
complementarity determining region (CDR) in the variable regions
such as the CDR3 and preferably all 6 CDRs have been replaced by
CDRs of an antibody of human origin having a desired specificity.
Optionally, the non-human constant region(s) of the antibody
has/have been replaced by (a) constant region(s) of a human
antibody. Methods for the production of humanized antibodies are
described in, e.g., EP-A1 0 239 400 and WO90/07861.
In a fifth aspect, the present invention provides a kit comprising
the above defined protein and/or the above defined peptide.
In a preferred embodiment, said kit further comprises one or more
of (a) a manual containing instructions for performing the method
of the first aspect; (b) an antibody in accordance with the fourth
aspect of the present invention.
The present invention, in a sixth aspect, provides the use of the
protein of the second aspect or of the peptide of the third aspect
for removing anti-KIR4.1 antibodies from blood or serum of an MS,
CIS or RIS patient or of a subject carrying a predisposition to
develop MS, CIS or RIS, or reducing the amount of said antibodies,
wherein said use is to be effected ex vivo.
Related thereto, the present invention provides an ex vivo method
of removing anti-KIR4.1 antibodies from a bodily fluid such as
blood or serum of an MS, CIS or RIS patient or of a subject
carrying a predisposition to develop MS, CIS or RIS, or reducing
the amount of said antibodies, wherein said use is to be effected
ex vivo, said method comprising bringing blood removed from a
subject into contact with a protein or peptide as defined
above.
These aspects relate to ex vivo applications, said ex vivo
applications aiming at a reduction of a number of autoantibodies or
a complete depletion thereof. Preferably, blood or serum of an MS
patient or of a subject carrying a predisposition to develop MS are
subjected to the ex vivo treatment. It is understood that said
bringing into contact is effected under conditions which allow
binding of autoantibodies, if present, to said protein or peptide.
In one embodiment, said conditions may be established by bringing
into contact blood or serum with a carrier or device according to
the invention, said carrier or device being further defined
below.
In a preferred embodiment of the ex vivo method according to the
invention, the blood or serum, after said bringing into contact, is
to be returned to the same subject.
In further preferred embodiments of the ex vivo use or the ex vivo
method of the invention, said protein or peptide is bound to a
carrier. Any carrier, including a solid carrier is envisaged.
Support or carrier materials commonly used in the art and
comprising glass, plastic, gold and silicon are envisaged for the
purpose of the present invention. Suitable coatings of the carrier
or support, if present, include poly-L-lysine- and
amino-silane-coatings as well as epoxy- and aldehyde-activated
surfaces. In a preferred embodiment, said carrier is the matrix of
a column. Suitable matrices are known in the art and may be
derivatized by the attachment of said peptide.
The present invention furthermore relates to a carrier with a
protein or peptide as defined above being immobilized thereon.
Related thereto, provided is also a device for removing anti-KIR4.1
antibodies from blood, said device comprising the carrier as
defined above.
In a preferred embodiment of the device, said device further
comprises an inlet and/or an outlet permitting to let blood or
serum of the subject flow across the filter and/or the blood or
serum being returned to the same subject.
As regards the embodiments characterized in this specification, in
particular in the claims, it is intended that each embodiment
mentioned in a dependent claim is combined with each embodiment of
each claim (independent or dependent) said dependent claim depends
from. For example, in case of an independent claim 1 reciting 3
alternatives A, B and C, a dependent claim 2 reciting 3
alternatives D, E and F and a claim 3 depending from claims 1 and 2
and reciting 3 alternatives G, H and I, it is to be understood that
the specification unambiguously discloses embodiments corresponding
to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E,
I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G;
B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C,
D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless
specifically mentioned otherwise.
Similarly, and also in those cases where independent and/or
dependent claims do not recite alternatives, it is understood that
if dependent claims refer back to a plurality of preceding claims,
any combination of subject-matter covered thereby is considered to
be explicitly disclosed. For example, in case of an independent
claim 1, a dependent claim 2 referring back to claim 1, and a
dependent claim 3 referring back to both claims 2 and 1, it follows
that the combination of the subject-matter of claims 3 and 1 is
clearly and unambiguously disclosed as is the combination of the
subject-matter of claims 3, 2 and 1. In case a further dependent
claim 4 is present which refers to any one of claims 1 to 3, it
follows that the combination of the subject-matter of claims 4 and
1, of claims 4, 2 and 1, of claims 4, 3 and 1, as well as of claims
4, 3, 2 and 1 is clearly and unambiguously disclosed.
The Figures show:
FIG. 1: KIR4.1 differs in white and gray matter by its molecular
weight. KIR4.1 was isolated from human brain and treated with
PNGase. Note that the differences in KIR4.1 molecular weight arise
from glycosylation.
FIG. 2: Binding of KIR4.1 specific antibodies.
FIG. 3: Sigma mouse mAb (A) targeting the C-terminal domain capture
a broad range of KIR4.1 proteins including highly glycosylated
KIR4.1. By contrast, the rat mAb (B) targeting the extracellular
domain of KIR4.1 and MS sera (C) pull predominantly the 38-42 kD
isoform of KIR4.1.
FIG. 4: (A-C) Costaining of the rat monoclonal antibody clone 20F9
with a commercial rabbit polyclonal anti-KIR4.1 antibody (Alomone,
APC-035). Notably, clone 20F9 binds to aextracellular domain of
KIR4.1 (peptide sequence AHGDLLELDPPANHT (SEQ ID NO: 2)), whereas
the polyclonal commercial antibody binds to a intracellular
C-terminal domain of KIR4.1 (peptide sequence KLEESLREQAEKEGSALSVR
(SEQ ID NO: 15)). Clone 20F9 binds to a similar epitope that is
bound by isolated anti-KIR4.1 IgG from serum obtained from MS
patients. Note that 20F9 immunohistochemistry (IHC) on subcortical
white matter control tissue displays predominantly a perinuclear
and cell membrane staining pattern. Most of the staining seems to
be restricted to cell bodies of oligodendrocytes (grey arrowheads)
as confirmed by costaining with the commercial anti-KIR4.1
antibody. Of note, some astrocytes (white arrows) show a
perinuclear staining with 20F9, however, it does not stain the
majority of astrocytic cell bodies and fibers as observed with the
commercial anti-KIR4.1 antibody. (D-F) Costaining of 20F9 with a
commercial rabbit polyclonal anti-Olig2 antibody (IBL) confirms
that 20F9 IHC predominantly has a perinuclear and cell membranes
staining pattern in oligodendrocytes (arrowheads). Olig2 is an
oligodendrocyte transcription factor. The anti-Olig2 antibody binds
specifically to nuclei of oligodendrocytes and oligodendrocyte
precursor cells. (G-I) Costaining of 20F9 with a mouse monoclonal
anti-NogoA antibody (clone 11C7, gift from M. Schwab, Zurich)
confirms that clone 20F9 binds to cytoplasms and cell membranes of
mature oligodendrocytes (arrowheads). The anti-NogoA antibody binds
specifically to cell bodies of mature oligodendrocytes. (J-L)
Costaining of 20F9 with a commercial mouse monoclonal anti-GFAP
antibody (Dako, 6F2) confirms that 20F9 staining is restricted to
oligodendrocytes (GFAP negative, arrowheads) and perinuclear areas
of a subset of astrocytes (white arrows, GFAP positive). Note that
astrocyte in the center is not stained with 20F9.
FIG. 5: ELISA with different KIR4.1 fractions isolated from
KIR4.1-his tag transfected HEK cells (HEK-KIR4.1-his). Note that
significant reactivity was only observed in the eluate, which
predominately contained the 38-42 kD variant of KIR4.1. Note that
human sera showed a similar reactivity as the rat monoclonal
antibody targeting the extracellular domain of KIR4.1. MS, Sera
from patients with MS; Ctr, Sera from patients with other
neurological diseases. P values are displayed for the comparison
between MS and Ctr.
The examples illustrate the invention.
EXAMPLE 1
Materials and Methods
For visualization of protein molecular weight in during
electrophoresis and Western Blotting, Blue.RTM. Plus2 Pre-Stained
Standard has been used.
Preparation of White Matter and Gray Matter Lysate
White matter or gray matter tissue were dissected from frozen human
brain and weighed for equal amount (2.4 g). Tissue was homogenized
using a glass tissue homogenizer in ice cold homogenization buffer
(0.32M sucrose, 10 mM HEPES pH 7.4, 2 mM EDTA) and protease
inhibitor cocktail (Sigma-Aldrich). The suspension was centrifuged
at 1000 g to pellet down the nuclear fraction. High speed
centrifugation and sucrose gradient method was used for the
enrichment of the membrane fraction. The enriched membrane pellet
was resuspended in HEPES solubilisation buffer (50 mM HEPES, 150
mMNaCl, 1.0% n-Dodecyl .beta.-D-maltoside and protease inhibitor
cocktail). The solubilised membrane protein extract were
investigated further either with direct immunoblotting or
immunoblotting of immunoprecipitated protein fractions. In case of
KIR4.1 transient transfected (reverse method) HEK293 cells or wild
type cell's total lysate were prepared with lysis buffer (50 mM
HEPES, 150 mMNaCl, 1.0% n-Dodecyl .beta.-D-maltoside and protease
inhibitor cocktail).
Immunoblotting
All denaturating Western blotting experiments were performed on
4-12% SDS gels (Invitrogen) with mixture of rabbit polyclonal
anti-human KIR4.1 antibodies (Millipore and Alomne lab) using pico
or femto ECL detection system (Thermo Scientific).
Immunoprecipitated or affinity purified samples were blotted after
N-deglycosylation or without deglycosylation. In case of native gel
western protein lysate were run on 4-16% bis-tris gels (Invitrogen)
in non-denaturating condition according to the manufacturer's
protocol.
IgG "Cross-Linking" on Protein-G Beads
Anti-Kir4.1 rat monoclonal antibody targeting the first
extracellular domain (mAb Kir4.1-ED), anti-Kir4.1C-terminal mouse
monoclonal, control antibodies or purified IgG from KIR4.1 reactive
serum were cross-linked on protein G beads as follow. 500 .mu.l
protein G beads (GE biosciences) were washed 3 time with PBS.
Washed protein G beads were incubated overnight at 4.degree. C.
with invert mixing (25-30 rpm) in 15 ml (30 .mu.g/ml) antibody-PBS
solution of either of antibodies. IgG bounded protein G beads were
washed with PBS 4 time and incubated with 5 ml of 50 mM borate
buffer for 2 min and the process was repeated for two times.
Further, IgGs bound protein G beads were incubated with
cross-linking solution (25 mg DMP in 4.5 ml of 50 mM borate buffer)
at 25 degree for 30 minutes. IgG cross-linked protein G beads were
washed with 5 ml of ethanolamine and then incubated further with 5
ml of ethanolamine for 1 hour at room temperature. Beads were
washed with 5 ml PBS and were stored as 50% slurry. These
cross-linked beads were used in 20-30 .mu.l amounts in all
immunoprecipitation experiments.
Immunoprecipitation
Protein lysate was pre-cleared with non-IgG bounded protein G beads
for non-specific binding. 30 .mu.l antibody "cross linked" protein
G beads were incubated with 300 .mu.g of total protein lysate in
300 .mu.l of IP buffer (20 mM tris, 150 mM NaCl, 1 mM EDTA) at
4.degree. C. for 3 hours. Beads were washed with wash buffer (IP
buffer supplemented with 0.1% n-Dodecyl .beta.-D-maltoside) two
times and further with IP buffer without EDTA two times. Captured
protein were eluted two times with 100 .mu.l soft elution buffer
(0.2 SDS, 0.1% Tween 20, 50 mM Tris pH 8.0) and further with 100
.mu.l of hard elution buffer (0.2 SDS, 50 mM Tris pH8.0) with an
incubation time of 7 minutes each at 25.degree. C. Each eluted
fraction was precipitated with the chloroform-methanol method.
Precipitated protein were blotted directly or after
N-deglycosylation.
N-Deglycosylation
Immunoprecipitated or affinity purified KIR4.1 protein were treated
with PNGase (New England Biolab) according manufacturer's
instruction. To determine the type of N-linked glycosylation PNGase
was used in combination with Endo-H according to manufacturer's
instruction.
Immunofluorescence
For immunofluorescence staining either freshly dissected CNS tissue
of human origin was snap frozen and embedded in tissue-tek O.C.T
(VWR Int., LLC, Radnor, Pa., USA) and Cryo-sectioning was performed
at -20.degree. C. to obtain 10 Pm sections or KIR4.1 transfected or
wild type HEK293 cells grown in Nunc pre-coated chamber slide were
used. After fixation with 100% ice cold methanol for 10 min (in
case of tissue) or 4 min (in case of cells), blocking steps were
performed with peroxidase, avidin and biotin blocking reagents
(Vector Laboratories Inc.) for 15 min each and with 10% goat, mouse
or rat serum in PBS-T (0.05% tween-20 in phosphate buffer saline pH
7.0) for 30 min. Sections were then incubated with diluted
anti-KIR4.1 rat monoclonal for extracellular loop or rabbit
polyclonal for C-terminal (10 ug/ml in PBS-T) overnight at
4.degree. C. After multiple washing steps, sections were incubated
with biotin-tagged secondary antibodies for 1 hr at room
temperature. Section were further incubated with Avidin-biotin
complex (Vector) for 1 hr, with 1 .mu.l of biotinylatedtyramide in
PBS with 8.8 mM of H.sub.2O.sub.2. All washing steps were performed
with PBS-T. Antibody binding was detected with AlexaFluor 488- or
AlexaFluor 555-labeled avidin. Nuclear staining was performed using
Gold antifade with DAPI (Invitrogen). In case of double staining
second primary antibody were used directly without avidin-biotin
complex or biotinylated secondary antibodies and probed alexa-488
or 555 labelled anti-goat, anti-rat or anti-rabbit antibodies.
Images were taken using a Zeiss Cell Observer microscope with an
AxioCamMRm camera (Carl Zeiss MicroImaging, Ltd., Gottingen,
Germany).
Cloning, Expression and Purification of KIR4.1
For recombinant KIR4.1 expression in HEK293 or Oli-Neu cells, a
full length cDNA encoding human KIR4.1 with C-terminal
hexa-histidine tag (his-tag) was synthesized from total human brain
mRNA (BD Biosciences, San Jose, Calif.) using 5'-GCG GCC GCA CCA
TGA CGT CAG TTG CCA AGG TGT ATT ACA GTC AG-3' (SEQ ID NO: 5) and
5'-CTC GAG TCA GTG GTGGTGGTGGTGGTG GAC ATT GCT GAT GCG CAC-3' (SEQ
ID NO: 6) as forward and reverse primers (his-tag encoding sequence
is underlined), respectively. Cloning into pcDNA 3.1(+)
(Invitrogen) was carried out using NotI and XhoI restriction sites
inserted via forward and reverse primers respectively to obtain
pcDNA 3.1(+)/KIR4.1 expression construct. HEK 293 cells were
transiently transfected (reverse method) with pcDNA 3.1(+)/KIR4.1
using lipofectamine 2000 transfection reagent (Invitrogen)
according to the manufacturer's instructions. At 6 hr
post-transfection medium was supplemented with 10% FCS and 300 mM
barium chloride. At 36 hours post-transfection cells were harvested
and washed twice with ice cold PBS. After counting, 2 billion cells
were subjected to lysis in 40 ml of 50 mM sodium phosphate buffer
pH 7.4 containing 150 mM sodium chloride, 1.0% n-Dodecyl
.beta.-D-maltoside, 500 units of Benzonase.RTM. nuclease (Sigma)
and 1.times.EDTA free protease inhibitor cocktail (Sigma). Cell
lysate was centrifuged at 20,000 rpm, using SS34 rotor on a Sorvall
RC6 plus centrifuge for 30 minutes at 4.degree. C. After
centrifugation, the supernatant (cleared lysate) was collected and
a total of 120-160 mg protein was loaded onto a purification column
containing 3 ml of HisPure.TM. cobalt resin (Pierce)
pre-equilibrated with 15 ml of binding buffer (lysis buffer with
0.1% Maltoside). Washing was carried out with 20 ml of washing
buffer (lysis buffer with 0.1% Maltoside). Elution of his tagged
protein fraction was carried out with 16 ml elution buffer (50 mM
sodium phosphate, 150 mM sodium chloride, 150 mM imidazole; pH
6.0). Eluted protein were further fractionated on Hiload 200 pg gel
filtration column (GE healthcare) and differentially fractionated
KIR4.1 oligomer with low glycosylation were used for ELISA.
KIR4.1 ELISA
KIR4.1 protein is isolated by cobalt bead-based affinity
purification (Thermo Scientific, Waltham, Mass.) according to
instruction in the method section of cloning, expression and
purification of KIR4.1. Affinity purified oligomeric KIR4.1 protein
is further fractionated and purified with Hi-load 200 pg gel
filtration column (GEBiosciences, Pittsburgh, Pa.). The quality of
the oligomeric protein reflects the glycosylation state of KIR4.1
protein expression in white matter (oligodendrocytes) and that may
be determined by running protein on 4% to 12% bis-tris gel
(Invitrogen, Carlsbad, Calif.) with reducing and nonreducing
conditions along with 4% to 16% blue native gel (Invitrogen).
Freshly purified protein is preferably used in all assays because
KIR4.1 protein is prone to form soluble aggregates at 4.degree. C.
or in freezing/thawing cycles. Plate coating is done at 4.degree.
C. overnight with 100 .mu.l of 7 .mu.g/ml of non-aggregated
oligomeric KIR4.1 in PBS. It is advisable to run titration of
protein coating concentration to know exact saturation point by
using 20F9 monoclonal antibody (generated against the first
extracellular domain of KIR4.1; competing with autoantibodies from
the sera of MS patients) as an indicator. Excess protein used above
saturation point is prone for aggregation in plate and creates
assay variability. After overnight coating plate is washed 3 time
with PBST (Phosphate Buffer Saline, 0.5% Tween 20) and blocked with
200 .mu.l of ultra blocking buffer BUF033 (Biorad) for 1 hour at
25.degree. C. Skimmed milk should be avoided as blocker because
differences in milk quality might have unpredictable effects on
epitope accessibility. After blocking, plates are washed for 3
times with PBST and should be incubated at 25.degree. C. with 100
.mu.l solution of serum or plasma diluted (1:100) in assay diluents
buffer BUF037B (Biorad) for 2-3 hour on an orbital shaker (rpm 90).
After incubation of sera, plates are washed 5 time with PBST before
probing with anti-human IgG secondary antibody (Sigma) diluted
(1:10000) in assay diluent buffer BUF037B. Platesare incubated at
25.degree. C. for 1 hour on an orbital shaker (rpm 90). Finally,
platesare washed for 5 time with PBST and assay should be develop
with 25.degree. C. equilibrated 100 .mu.l TMB solution for 22-25
minutes. Reaction is stopped with 50 .mu.l of 2N H.sub.2SO.sub.4.
Samples are preferably run in triplicate to determine intra-assay
variation. All sera and the sera to determine the cut-off value
should be measure in the same assay. None of the samples used in
the assays should be ever thawed or refrozen. For best performance
average control serum background optical density (OD) should be
around 0.5 with maximum limit of 0.8. Maximum inter-assay variation
acceptable for the best quality of assay is a CV of less than 8%
and intra-assay variation of less than 5%. The cut-off value should
be determined for each assay by the OD obtained from the same set
of healthy control (HC) donors. The assay is sensitive for serum
quality. The average OD as well as the intra and inter assay
variation obtained with KIR4.1 antibody negative MS sera should
reflect those of sera from patients with other neurological
diseases (OND). The assay is preferably with at least 3 different
protein preparations to determine inter-assay variability.
EXAMPLE 2
Results
Differential Glycosylation of KIR4.1
KIR4.1 has an N-glycosylation site, which is located in the larger
extracellular domain and can undergo differential glycosylation
(see FIG. 1). Analysis of KIR.1 protein from subcortical white and
cortical gray matter revealed differences in the molecular weight
of the protein. In white matter a 38-42 kD protein, in gray matter
a 49-55 kD KIR4.1 protein is dominant. Interestingly, the 49-55 kD
KIR4.1 is also dominant in glioma tissue. Deglycosylation with
PNGase, which specifically remove N-glycosylation, revealed in both
instances a 34 kD protein corresponding to the MW of deglycosylated
KIR4.1. This suggests that KIR4.1 is differentially N-glycosylated
in the CNS (see FIG. 1).
Antibody competing with the Autoantibody from MS Patients
To further address this point we generated a rat monoclonal
antibody (20F9) against the first extracellular domain of KIR4.1
(FIG. 2). The 20F9 antibody is an example of the antibody in
accordance with the fourth aspect of this invention.
The 20F9 antibody precipitates predominantly the lower glycosylated
38-42 kD KIR4.1. In contrast to monoclonal antibodies targeting the
C-terminus of KIR4.1, this antibody does not pull the highly
glycosylated 49-55 kD KIR4.1 protein. Interestingly, the same
binding pattern is observed with KIR4.1 specific antibodies from MS
sera (see FIG. 3). These findings suggest that a higher
glycosylation of the antigenic epitope might have a steric effect
on the antigen-antibody interaction and possibly inhibit antibody
binding.
Oligodendrocyte-Specific KIR4.1 Glycosylation
We carried out stainings of human brain tissue with the 20F9
antibody (FIG. 4). This antibody stains predominantly
oligodendrocytes and to a lesser extent astrocytes. Interestingly,
the staining pattern of this antibody is significantly different
from the staining pattern of monoclonal antibodies targeting the
C-terminus of KIR4.1. While the latter antibodies stain astrocytes
and oligodendrocytes to a similar extent, 20F9 preferentially
stains oligodendrocytes and fails to stain astrocytic fibers
suggesting that the glycosylation state of KIR4.1 differs between
astrocytes and oligodendrocytes.
To demonstrate the impact of different glycosylation for antibody
binding, we fractionated the recombinant KIR4.1 protein isolated
from HEK cells and tested MS sera for reactivity with the different
fractions. KIR4.1 antibodies comprised in the serum of MS patients
reacted with fraction 1 in which the 38-42 kD protein was enriched
but failed to react with fraction 2 that contained predominantly
heavily glycosylated KIR4.1 (FIG. 5). Similarly the 20F9 antibody
reacted strongly with fraction 1 but much less with fraction 2
although the same protein concentration was used. In line with this
finding, MS sera captured 38-42 kD KIR4.1 protein but failed to
capture heavily glycosylated KIR4.1 from WT and KIR4.1 transfected
HEK cells (FIG. 4).
CONCLUSION
KIR4.1 occurs in several glycosylation states ranging from no
glycosylation to heavy glycosylation. Oligodendrocytes express low
glycosylated KIR4.1.
The antigenic epitope in the extracellular loop of KIR4.1 contains
an N-glycosylation motif, which is prone to complex glycosylation.
The extent of glycosylation is depending on cell type and
stage.
By generating a monoclonal antibody targeting the same
extracellular domain of human KIR4.1 (clone 20F9), which is
reactive to human serum IgG, we observed that the level of
glycosylation of the extracellular domain has a major impact on
antibody binding. 20F9 antibody binds to KIR4.1 only when the
protein is weakly or not glycosylated. Heavy glycosylation
abrogates binding.
IgG from MS sera behave similar to 20F9 antibody. The antibodies
bind to weakly or non-glycosylated KIR4.1 (corresponding to the
38-42 kD variants expressed in oligodendrocytes) but not heavily
glycosylated KIR4.1 protein. Assays that do not display the 38-42
KIR4.1 variant may not be able to detect the antibody in MS sera
and may therefore provide false negative results.
SEQUENCE LISTINGS
1
1715PRTArtificial SequenceKIR4.1 fragment 1Asp Pro Pro Ala Asn1
5215PRTArtificial SequenceKIR4.1 fragment 2Ala His Gly Asp Leu Leu
Glu Leu Asp Pro Pro Ala Asn His Thr1 5 10 153379PRTHomo sapiens
3Met Thr Ser Val Ala Lys Val Tyr Tyr Ser Gln Thr Thr Gln Thr Glu1 5
10 15Ser Arg Pro Leu Met Gly Pro Gly Ile Arg Arg Arg Arg Val Leu
Thr 20 25 30Lys Asp Gly Arg Ser Asn Val Arg Met Glu His Ile Ala Asp
Lys Arg 35 40 45Phe Leu Tyr Leu Lys Asp Leu Trp Thr Thr Phe Ile Asp
Met Gln Trp 50 55 60Arg Tyr Lys Leu Leu Leu Phe Ser Ala Thr Phe Ala
Gly Thr Trp Phe65 70 75 80Leu Phe Gly Val Val Trp Tyr Leu Val Ala
Val Ala His Gly Asp Leu 85 90 95Leu Glu Leu Asp Pro Pro Ala Asn His
Thr Pro Cys Val Val Gln Val 100 105 110His Thr Leu Thr Gly Ala Phe
Leu Phe Ser Leu Glu Ser Gln Thr Thr 115 120 125Ile Gly Tyr Gly Phe
Arg Tyr Ile Ser Glu Glu Cys Pro Leu Ala Ile 130 135 140Val Leu Leu
Ile Ala Gln Leu Val Leu Thr Thr Ile Leu Glu Ile Phe145 150 155
160Ile Thr Gly Thr Phe Leu Ala Lys Ile Ala Arg Pro Lys Lys Arg Ala
165 170 175Glu Thr Ile Arg Phe Ser Gln His Ala Val Val Ala Ser His
Asn Gly 180 185 190Lys Pro Cys Leu Met Ile Arg Val Ala Asn Met Arg
Lys Ser Leu Leu 195 200 205Ile Gly Cys Gln Val Thr Gly Lys Leu Leu
Gln Thr His Gln Thr Lys 210 215 220Glu Gly Glu Asn Ile Arg Leu Asn
Gln Val Asn Val Thr Phe Gln Val225 230 235 240Asp Thr Ala Ser Asp
Ser Pro Phe Leu Ile Leu Pro Leu Thr Phe Tyr 245 250 255His Val Val
Asp Glu Thr Ser Pro Leu Lys Asp Leu Pro Leu Arg Ser 260 265 270Gly
Glu Gly Asp Phe Glu Leu Val Leu Ile Leu Ser Gly Thr Val Glu 275 280
285Ser Thr Ser Ala Thr Cys Gln Val Arg Thr Ser Tyr Leu Pro Glu Glu
290 295 300Ile Leu Trp Gly Tyr Glu Phe Thr Pro Ala Ile Ser Leu Ser
Ala Ser305 310 315 320Gly Lys Tyr Ile Ala Asp Phe Ser Leu Phe Asp
Gln Val Val Lys Val 325 330 335Ala Ser Pro Ser Gly Leu Arg Asp Ser
Thr Val Arg Tyr Gly Asp Pro 340 345 350Glu Lys Leu Lys Leu Glu Glu
Ser Leu Arg Glu Gln Ala Glu Lys Glu 355 360 365Gly Ser Ala Leu Ser
Val Arg Ile Ser Asn Val 370 375438PRTHomo sapiens 4Gly Val Val Trp
Tyr Leu Val Ala Val Ala His Gly Asp Leu Leu Glu1 5 10 15Leu Asp Pro
Pro Ala Asn His Thr Pro Cys Val Val Gln Val His Thr 20 25 30Leu Thr
Gly Ala Phe Leu 35544DNAArtificial
Sequencesource1..44/mol_type="unassigned DNA" /note="Primer"
/organism="Artificial Sequence" 5gcggccgcac catgacgtca gttgccaagg
tgtattacag tcag 44645DNAArtificial
Sequencesource1..45/mol_type="unassigned DNA" /note="Primer"
/organism="Artificial Sequence" 6ctcgagtcag tggtggtggt ggtggtggac
attgctgatg cgcac 4576PRTArtificial SequenceKIR4.1 fragment 7Leu Asp
Pro Pro Ala Asn1 587PRTArtificial SequenceKIR4.1 fragment 8Glu Leu
Asp Pro Pro Ala Asn1 598PRTArtificial SequenceKIR4.1 fragment 9Leu
Glu Leu Asp Pro Pro Ala Asn1 5109PRTArtificial SequenceKIR4.1
fragment 10Leu Leu Glu Leu Asp Pro Pro Ala Asn1 51110PRTArtificial
SequenceKIR4.1 fragment 11Asp Leu Leu Glu Leu Asp Pro Pro Ala Asn1
5 101211PRTArtificial SequenceKIR4.1 fragment 12Gly Asp Leu Leu Glu
Leu Asp Pro Pro Ala Asn1 5 101312PRTArtificial SequenceKIR4.1
fragment 13His Gly Asp Leu Leu Glu Leu Asp Pro Pro Ala Asn1 5
101413PRTArtificial SequenceKIR4.1 fragment 14Ala His Gly Asp Leu
Leu Glu Leu Asp Pro Pro Ala Asn1 5 101520PRTArtificial
SequenceKIR4.1 fragment 15Lys Leu Glu Glu Ser Leu Arg Glu Gln Ala
Glu Lys Glu Gly Ser Ala1 5 10 15Leu Ser Val Arg 20166PRTArtificial
SequenceKIR4.1 fragment 16Pro Pro Ala Asn His Thr1
5174PRTArtificial SequenceKIR4.1 fragment 17Pro Pro Ala Asn1
* * * * *